Les J. Szabo

Obligate biotrophy features unraveled by the genomic analysis of rust fungi

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

Obligate Biotrophy Features Unraveled by the Genomic Analysis of the Rust Fungi, Melampsora larici-populina and Puccinia graminis f. sp. tritici

Rust fungi are some of the most devastating pathogens of crop plants. They are obligate biotrophs, which extract nutrients only from living plant tissues and cannot grow apart from their hosts. Their lifestyle has slowed the dissection of molecular mechanisms underlying host invasion and avoidance or suppression of plant innate immunity. We sequenced the 101-Mb genome of Melampsora larici-populina, the causal agent of poplar leaf rust, and the 89-Mb genome of Puccinia graminis f. sp. tritici, the causal agent of wheat and barley stem rust. We then compared the 16,399 predicted proteins of M. larici-populina with the 17,773 predicted proteins of P. graminis f. sp tritici. Genomic features related to their obligate biotrophic lifestyle include expanded lineage-specific gene families, a large repertoire of effector-like small secreted proteins, impaired nitrogen and sulfur assimilation pathways, and expanded families of amino acid and oligopeptide membrane transporters. The dramatic up-regulation of transcripts coding for small secreted proteins, secreted hydrolytic enzymes, and transporters in planta suggests that they play a role in host infection and nutrient acquisition. Some of these genomic hallmarks are mirrored in the genomes of other microbial eukaryotes that have independently evolved to infect plants, indicating convergent adaptation to a biotrophic existence inside plant cells.

Detection of virulence to resistance gene Sr24 within race TTKS of Puccinia graminis f. sp. tritici

The stem rust resistance gene Sr24 is effective against most races of Puccinia graminis f. sp. tritici, including race TTKS (syn. Ug99), and is used widely in commercial wheat cultivars worldwide. In 2006, susceptible infection responses were observed on wheat lines and cultivars carrying Sr24 in a field stem rust screening nursery at Njoro, Kenya. We derived 28 single-pustule isolates from stem rust samples collected from the 2006 Njoro nursery. The isolates were evaluated for virulence on 16 North American stem rust differential lines; on wheat lines carrying Sr24, Sr31, Sr38, and SrMcN; and on a wheat cultivar with a combination of Sr24 and Sr31. All isolates were identified as race TTKS with additional virulence on Sr31 and Sr38. These isolates were divided into two groups: group A (seven isolates and the two control isolates), producing a low infection type, and group B (21 isolates), producing a high infection type on Sr24, respectively. Isolates of group B represented a new variant of race TTKS with virulence to Sr24. Eighteen simple sequence repeat (SSR) markers were used to examine the genetic relationship between these two groups of isolates in race TTKS and five North American races (MCCF, QCCQ, RCRS, RTHS, and TPMK) that are representative of distinct lineage groups. All isolates of race TTKS shared an identical SSR genotype and were clearly different from North American races. The virulence and SSR data indicated that the new variant of race TTKS with Sr24 virulence likely has arisen via mutation within the TTKS genetic lineage. We propose to revise the North American stem rust nomenclature system by the addition of four genes (Sr24, Sr31, Sr38, and SrMcN) as the fifth set. This revision recognizes the virulence on Sr31 and differentiates isolates within race TTKS into two separate races: TTKSK and TTKST, with avirulence and virulence on Sr24, respectively. The occurrence of race TTKST with combined virulence on Sr24 and Sr31 has substantially increased the vulnerability of wheat to stem rust worldwide.

Century-old mystery of Puccinia striiformis life history solved with the identification of Berberis as an alternate host.

The life history of Puccinia striiformis remains a mystery because the alternate host has never been identified. Inoculation of grasses using aeciospores from naturally infected Berberis chinensis and B. koreana resulted in infection on Poa pratensis, producing uredinia typical of stripe rust caused by P. striiformis. Analyses using real-time polymerase chain reaction and DNA sequence confirmed the rust fungus as P. striiformis. Pycnia and aecia were produced on B. chinensis, B. holstii, B. koreana, and B. vulgaris after inoculation using germinating telia of P. striiformis f. sp. tritici. Wheat inoculated with aeciospores from B. chinensis resulted in uredinia, which demonstrated that Berberis spp. also serve as alternate hosts for the wheat stripe rust pathogen. The elucidation of the complete life history for P. striiformis f. sp. tritici will provide a powerful tool to rapidly advance our knowledge of the genetics of this rust fungus, and will lead to the development of improved strategies for a better control of stripe rust.

Stem rust of small grains and grasses caused by Puccinia graminis

UNLABELLED SUMMARY Stem rust has been a serious disease of wheat, barley, oat and rye, as well as various important grasses including timothy, tall fescue and perennial ryegrass. The stem rust fungus, Puccinia graminis, is functionally an obligate biotroph. Although the fungus can be cultured with difficulty on artificial media, cultures grow slowly and upon subculturing they develop abnormal ploidy levels and lose their ability to infect host plants [Bushnell and Bosacker (1982) Can. J. Bot. 60, 1827-1836]. P. graminis is a typical heteroecious rust fungus with the full complement of five distinct spore stages that occur during asexual reproduction on its gramineous hosts and sexual reproduction that begins in the resting spore stage and culminates on the alternate host, barberry (Berberis spp.). There appears to be little polymorphism for resistance/susceptibility in Berberis species, but complex polymorphisms of resistance/susceptibility and matching virulence/avirulence exist in gene-for-gene relationships between small grain species and the forms of P. graminis that infect them. TAXONOMY Puccinia graminis is a rust fungus in the phylum Basidiomycota, class Urediniomycetes, order Uredinales, and family Pucciniaceae, which contains 17 genera and approximately 4121 species, of which the majority are in the genus Puccinia[Kirk et al. (2001) Ainsworth and Bisbys Dictionary of the Fungi. Wallingford, UK: CAB International]. Various subdivisions of P. graminis into subspecies, varieties and formae speciales have been proposed based on spore size and host range. Crossing studies and DNA sequence comparisons support the separation of at least two subspecies, but not the proposed separation based on spore size. HOST RANGE The host range of P. graminis is very broad compared with that of most Puccinia spp.; it includes at least 365 species of cereals and grasses in 54 genera [Anikster (1984) The Cereal Rusts. Orlando, FL: Academic Press, pp. 115-130]. Wheat stem rust, P. graminis f. sp. tritici, was shown to infect 74 species in 34 genera in artificial inoculations of seedlings, but only 28 of those species belonging to eight genera were known to be natural hosts of the fungus. Other formae speciales of P. graminis have narrower host ranges than P. graminis f. sp. tritici. Disease symptoms: Infections in cereals or grasses occur mainly on stems and leaf sheaths, but occasionally they may be found on leaf blades and glumes as well. The first macroscopic symptom is usually a small chlorotic fleck, which appears a few days after infection. About 8-10 days after infection, a pustule several millimetres long and a few millimetres wide is formed by rupture of the host epidermis from pressure of a mass of brick-red urediniospores produced in the infection. These uredinial pustules are generally linear or diamond shaped and may enlarge up to 10 mm long. The powdery masses of urediniospores appear similar to rust spots on a weathered iron surface. With age, the infection ceases production of brick-red urediniospores and produces a layer of black teliospores in their place, causing the stems of heavily infected plants to appear blackened late in the season.

Detection of virulence to resistance gene Sr36 within the TTKS race lineage of Puccinia graminis f. sp. tritici

The stem rust resistance gene Sr36 confers a near-immune resistance reaction to many races of Puccinia graminis f. sp. tritici and is highly effective against race TTKSK (syn. Ug99), which possesses unusually broad virulence combinations. Because this gene is widely used in United States soft winter wheat germplasm and cultivars, it has been considered to be an important source of resistance to TTKSK. In 2007, moderately susceptible infection responses were observed on wheat lines and cultivars carrying Sr36 in a field screening nursery for stem rust at Njoro, Kenya. We derived 18 single-pustule isolates from stem rust samples collected from the 2007 Njoro nursery. The isolates were evaluated for virulence on 20 North American stem rust differential lines and on wheat lines and cultivars carrying Sr36, Sr31+Sr36, and Sr24+Sr31. Of the 18 isolates, 10 produced infection types 3+ to 4 on line W2691SrTt-1 (monogenic for Sr36) and other lines that carry Sr36 and belonged to a new virulence phenotype that was not detected in previous years. These isolates were identified as race TTTSK. The remaining eight isolates were identified as races TTKSK (five isolates) and TTKST (three isolates), with avirulence and virulence, respectively, to Sr24. Thirteen simple sequence repeat (SSR) markers were used to examine the genetic relationships among the three races in the TTKS lineage. All isolates in the lineage shared an identical SSR genotype and were clearly different from North American races. In all, 16 wheat cultivars and 60 elite breeding lines, postulated to possess Sr36, were susceptible to race TTTSK. The occurrence of race TTTSK with combined virulence on Sr31 and Sr36 has further broadened the virulence spectrum of the TTKS lineage and rendered an important source of resistance ineffective.

Using Hierarchical Clustering of Secreted Protein Families to Classify and Rank Candidate Effectors of Rust Fungi

Rust fungi are obligate biotrophic pathogens that cause considerable damage on crop plants. Puccinia graminis f. sp. tritici, the causal agent of wheat stem rust, and Melampsora larici-populina, the poplar leaf rust pathogen, have strong deleterious impacts on wheat and poplar wood production, respectively. Filamentous pathogens such as rust fungi secrete molecules called disease effectors that act as modulators of host cell physiology and can suppress or trigger host immunity. Current knowledge on effectors from other filamentous plant pathogens can be exploited for the characterisation of effectors in the genome of recently sequenced rust fungi. We designed a comprehensive in silico analysis pipeline to identify the putative effector repertoire from the genome of two plant pathogenic rust fungi. The pipeline is based on the observation that known effector proteins from filamentous pathogens have at least one of the following properties: (i) contain a secretion signal, (ii) are encoded by in planta induced genes, (iii) have similarity to haustorial proteins, (iv) are small and cysteine rich, (v) contain a known effector motif or a nuclear localization signal, (vi) are encoded by genes with long intergenic regions, (vii) contain internal repeats, and (viii) do not contain PFAM domains, except those associated with pathogenicity. We used Markov clustering and hierarchical clustering to classify protein families of rust pathogens and rank them according to their likelihood of being effectors. Using this approach, we identified eight families of candidate effectors that we consider of high value for functional characterization. This study revealed a diverse set of candidate effectors, including families of haustorial expressed secreted proteins and small cysteine-rich proteins. This comprehensive classification of candidate effectors from these devastating rust pathogens is an initial step towards probing plant germplasm for novel resistance components.

Phylogenetic relationships among some cercosporoid anamorphs of Mycosphaerella based on rDNA sequence analysis

Partial rDNA sequences were obtained from 26 isolates representing species of Cercospora, Passalora, Paracercospora, Pseudocercospora, Ramulispora, Pseudocercosporella and Mycocentrospora. The combined internal transcribed spacers (ITS) including the 5.8S rRNA gene and 5′ end of the 25S gene (primer pairs F63/R635) on rDNA were amplified using PCR and sequenced directly. The ITS regions including the 5.8S varied in length from 502 to 595 bp. The F63/R635 region varied from 508 to 519 bp among isolates sequenced. Reconstructed phylogenies inferred from both regions had highly similar topologies for the taxa examined. Phylogenetic analysis of the sequences resulted in four well-supported clades corresponding to Cercospora, Paracercospora/Pseudocercospora, Passalora and Ramulispora, with bootstrap values greater than 92% for each clade. Based on the results of the analysis, a new combination for Pseudocercosporella aestiva is proposed in Ramulispora, and Paracercospora is reduced to synonymy with Pseudocercospora.

Hidden robbers: The role of fungal haustoria in parasitism of plants

Biotrophic fungi have developed a range of “life styles” in their relationship with plants from the mutualistic to the parasitic. Vesicular-arbuscular mycorrhizal fungi form mutualistic relationships with the roots of their plant hosts, in which the fungus obtains sugars from the plant and provides phosphates and other minerals in return. At the other extreme, powdery mildew and rust fungi form an obligately parasitic relationship in which the host plant becomes a source for sugars, amino acids, and other nutrients. These parasites develop a specialized organ, the haustorium (Fig. 1) within plant cells for transfer of nutrients from host cell to fungal thallus. The haustorium is assumed to have a key role in the ability of these parasites to compete with the developing plant for photoassimilates and other nutrients but basic questions remain regarding the function of the haustorium. These include: What are the major nutrients transported? What mechanisms are involved in the transport? How do individual components of the haustorium–host cell interface contribute to nutrient flow? And overall, how does haustorial function relate to the biotrophic relationship between host and parasite? The paper by Voegele et al. (1) in this issue of PNAS provides an important advance by characterizing a sugar transporter located at the haustorium–host interface.

Innate nonhost immunity in barley to different heterologous rust fungi is controlled by sets of resistance genes with different and overlapping specificities

We developed an evolutionary relevant model system, barley-Puccinia [corrected] rust fungi, to study the inheritance and specificity of plant factors that determine to what extent innate nonhost immunity can be suppressed. A mapping population was developed from a cross between an experimental barley line (SusPtrit) [corrected] with exceptional susceptibility to several heterologous [corrected] (nonhost) rust fungi and regular, immune, cv. Vada [corrected] Seedlings were inoculated with five heterologous [corrected] and two homologous (host) species of rust fungi. Resistance segregated quantitatively for each of the rust fungi. In total, 18 chromosomal regions were implicated. For each rust species, a different set of genes was effective. Of the 18 chromosomal regions, 11 were significantly effective to only one rust species and 7 were effective to more than one rust species, implying genetic linkage or pleiotropy. One resistance (R) gene for hypersensitive resistance to Puccinia hordei-secalini was mapped, suggesting occasional contribution of R genes to nonhost resistance in barley. Quantitative trait loci (QTLs) with effects to multiple rust fungi did not tend to be particularly effective to rust species that were phylogenetically related, as determined from their internal transcribed spacer sequence. We suggest that the QTLs described here play a role as specific and quantitative recognition factors that are specifically negated by the rust to successfully suppress innate immunity.